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Article

The Diversity of Culture-Dependent Gram-Negative Rhizobacteria Associated with Manihot esculenta Crantz Plants Subjected to Water-Deficit Stress

by
Tatiana Zapata
,
Diana Marcela Galindo
,
Alba Rocío Corrales-Ducuara
and
Iván Darío Ocampo-Ibáñez
*
Research Group of Microbiology, Industry and Environment, Faculty of Basic Sciences, Universidad Santiago de Cali, Cali 760035, Colombia
*
Author to whom correspondence should be addressed.
Diversity 2021, 13(8), 366; https://doi.org/10.3390/d13080366
Submission received: 1 July 2021 / Revised: 27 July 2021 / Accepted: 31 July 2021 / Published: 7 August 2021
(This article belongs to the Special Issue Microbial Diversity Associated with Photosynthetic Organisms)
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Abstract

:
There is a lack of studies on the root-associated bacterial microbiome of cassava plants. The identification and characterization of rhizobacteria can contribute to understanding the adaptation of the agriculturally important crop plants to abiotic stress. Rhizobacteria play a significant role in plants, as they can alleviate the drought stress by various mechanisms that enhance the plant growth under these stressor conditions. In this study, Gram-negative bacterial strains from the plant rhizosphere of cassava Manihot esculenta Crantz CIAT MCOL1734 variety subjected to water deprivation were isolated, characterized according to their morphological properties, and then identified by VITEK® 2. An increase in the diversity, abundance, and species richness of Gram-negative rhizobacterial community was found in cassava plants subjected to water-deficit stress. In total, 58 rhizobacterial strains were isolated from cassava plants. The identification process found that the bacteria belonged to 12 genera: Achromobacter, Acinetobacter, Aeromonas, Buttiauxella, Cronobacter, Klebsiella, Ochrobactrum, Pluralibacter, Pseudomonas, Rhizobium, Serratia, and Sphingomonas. Interestingly, Pseudomonas luteola and Ocrhobactrum anthropi were rhizobacteria isolated exclusively from plants submitted to drought conditions. The cassava roots constitute a great reservoir of Gram-negative bacteria with a remarkable potential for biotechnological application to improve the drought tolerance of plant crops under water-deficit conditions.

1. Introduction

Cassava (Manihot esculenta Crantz) is widely grown in subtropical and tropical areas worldwide, including Africa, Asia, Latin America, and the Caribbean [1,2]. In these regions, the cassava root is one of the most essential sources of calories after rice and corn and provides staple food to an estimated 800 million people [1]. In addition, the top biomass including leaves and immature stems may be used as the cassava hay for animal feeding [2,3].
The cassava crops are well adapted to several agroecological conditions; even its potential to adapt well to climate change is a factor that favors the increased production of cassava [1]. However, several factors, such as salinity and drought, may cause crop yield losses of more than 50% worldwide [4,5]. Despite these reasons, cassava can withstand relatively prolonged periods of drought during the first three months; however, after planting, the cassava crop is very sensitive to the soil water deficit [1,5,6,7]. At any time during this early establishment period, the water-deficit stress may affect the root system development, significantly reducing the growth of roots and shoots and, subsequently, the storage roots [1,8]. After this time, a water-deficit causes turgor loss, diminished water potential, disruption of membrane integrity along with protein denaturation, and stomatal closure, which may decline the rate of photosynthesis [9,10]. After planting, stakes only sprout and grow well when the soil moisture content is at least 30% of the field capacity. Once established, cassava can grow in dry areas [6,7,11,12]. Because the greatest potential for an increased yield of cassava production is in the rainfed areas and drylands of subtropical and tropical regions [1,13], the breeding of several higher-yielding varieties with improved root quality and tolerance to drought have been produced to be used in those areas [14,15,16]. The most common breeding objectives of these varieties include physiological mechanisms, such as delay in flowering time, reduction in transpiration rate, increased production of primary and secondary metabolites, hormone-mediated regulation of vegetative development, and an increased production of abscisic acid (ABA) that act as a mediator in the response to drought stress [17,18].
In addition to the breeding of tolerant varieties, there are natural strategies to rescue the plant growth in abiotic stressful conditions, for example, the activity of microorganisms associated with the rhizosphere [10,19,20]. Bacteria are the most abundant group in the rhizosphere, and they are referred to as plant-growth-promoting rhizobacteria (PGPR) [10,19,20]. Rhizobacteria are considered a bulwark because they colonize plant roots and possess a tremendous potential in promoting plant growth to ensure its survival under stressful conditions [10]. In particular, PGPRs have an inherent capacity to cope with abiotic stresses; for instance, they are able to boost plant tolerance to drought stress. Through a process called rhizobacterial-induced drought endurance and resilience, the PGPRs can mitigate the impact of water deprivation on plants [10]. This process includes physiological and biochemical modulations in plants, such as changes in the antioxidant defense and modifications in the production and content of phytohormones, such as indole-3-acetic acid (IAA), ABA, gibberellic acid, and cytokinin (CK) [10,21,22,23]. In addition, to ensure the survival of plants under drought-stressed conditions, the rhizobacteria can produce osmolytes, bacterial exopolysaccharides, heat-shock proteins, and volatile organic compounds, which may contribute to the drought tolerance of plants [10,24,25]. A large numbers of rhizobacteria strains belonging to several genera, including Achromobacter, Acinetobacter, Azotobacter, Bacillus, Pseudomonas, Rhizobium, and Serratia, have been identified from soils and rhizospheres of diverse plant species [26,27]. Several studies have demonstrated that the inoculation of plants with rhizobacteria is a biotechnological tool that may improve the productivity of crops under abiotic stress conditions [10,23]. When these procedures were implemented particularly in crops under a drought stress environment, an enhancement in the root system was observed, which improved the plant’s ability to uptake water and enhanced the crop productivity [10,23].
Cassava plants have a wide range of associated microorganisms that are distributed throughout the body of plants, including leaves, stems, and roots [28,29]. Previously, several studies characterized the bacterial diversity of the cassava rhizosphere and evaluated the treatment effects with PGPR on plant growth [30,31]. In this respect, a wide diversity of rhizobacteria associated with cassava has been identified including strains belonging to the genera Bacillus, Micrococcus, Pseudomonas, Enterobacter, Klebsiella, and Serratia, which were isolated from several cassava varieties and cultivars [31,32]. When cassava stem cuttings were in vitro treated with the isolates of these genera and then planted, the production of IAA and ammonia, phosphate solubilization, and increased iron levels in the leaf were induced, suggesting that rhizobacterial isolates from cassava may have potential as plant-growth promoters [30,31,33]. Despite these studies, very few studies have studied the rhizobacteria in cassava under abiotic stress conditions, and the structure and diversity of the bacterial communities of the rhizosphere of cassava plants under these conditions are not well known [34]. Recently, several studies have shown that the microbial composition of the rhizosphere in several plant species may be affected under abiotic stress conditions, for example, increasing the relative abundance of PGPRs by root colonization under drought stress [27,35,36,37]. In this study, we isolated bacterial strains from the rhizosphere of cassava (M. esculenta Crantz) subjected to water deprivation. Once isolated, the strains were characterized according to their morphological properties and then identified. The bacterial species here identified were reported as PGPR in previous studies.

2. Materials and Methods

2.1. Drought Stress Assays

Experiments were conducted under glasshouse conditions, such as temperature of 28–30 °C and relative humidity of 50 ± 80%, at the International Center for Tropical Agriculture (CIAT) (Palmira, Valle del Cauca, Colombia, 03°31′ N, 76°18′ W; a 1001 m.a.s.l.) between 1 August and 1 September 2018. All experiments were performed following a completely randomized design. Seven plants of CIAT MCOL1734 cassava variety [38] at vegetative stage were used to perform every assay. Five cassava plants (cp-ds-1, cp-ds-2, cp-ds-3, cp-ds-4, and cp-ds-5) were exposed to drought stress by experimentally manipulating irrigation to artificially impose water deficit for 20 days [39,40,41]. During this time, two cassava plants (cp-c-1 and cp-c-2) were exposed to daily irrigation and used as controls.

2.2. Strain Isolation

Taproots and adventitious roots were collected from cp-ds and cp-c plants at the beginning of assays (T0) and after 20 days of drought stress assay (T20). To isolate rhizobacteria only from the rhizoplane and endorhizosphere, the adhering soil particles were removed by washing the roots with abundant sterile distilled water. About 1 g of roots were collected and then macerated in a mortar. Next, 9 mL of Tween 80 solution (Milli-Q distilled water) was added into it to make a 10× dilution. Then, 1 mL of this mixture was used for serial 1:10 dilutions using buffered peptone water. From about 10−2 to 10−7 dilutions were plated in triplicates on the following media: nutrient agar (NA) (Sigma-Aldrich), Ashby’s mannitol agar (AMA) (HIMEDIA), King Agar B (KAB) (Sigma-Aldrich), and eosin methylene blue (EMB) Agar (Becton Dickinson). They were then incubated at 37 °C ± 2 °C for 18–24 h [42,43,44]. Distinct colonies obtained from agar plates were selected and then purified by subculturing.

2.3. Strain Characterization and Identification

Distinct bacterial colonies purified by subculturing were initially examined for colony characteristics (appearance, elevation, margin, pigmentation, shape, size, and texture) [45] and cellular characteristics (cell shape, gram testing, and production of spores) via microscopy [45,46]. In addition, the identification of bacterial species was based on biochemical testing. Catalase and oxidase tests were initially performed; then, species identification was performed using the automated VITEK® 2 system, (bioMerieux, 9.02, Marcy l’Etoile, France) with the VITEK® 2 Gram-negative identification card, which is based on established biochemical methods and substrates that evaluate the use of carbon and enzymatic activities (Reference 21341, bioMerieux, Marcy l’Etoile, France).

2.4. Statistical Analysis

Species diversity indices, such as the Shannon diversity index [47] and the Pielou evenness index [48], were determined to analyze bacterial communities in each group of cassava plants; in addition, bacterial species richness and abundance were estimated. Diversity indices, richness, and abundance were calculated from standardized profiles of individual cassava samples using the number of isolates for each identified bacterial species. The Shannon diversity index was calculated as follows:
H ′ = − ∑ ( p i ) ( ln   p i )
The Pielou evenness index was derived from the Shannon diversity index and was calculated as follows:
J ′ = H ′ / H ′ m a x
were H′ is the Shannon diversity index, pi is the relative impo1rtance value of species i, J′ is the Pielou evenness index, H′max = ln(S), and S is the total number of species in ith plot.
Statistically significant differences of the bacterial diversity in cp-c and cp-ds, comparing independently T0 and T20, were analyzed and compared using Shannon diversity index through the Hutcheson t-test [49]. p-values ≤ 0.05 were considered statistically significant. All calculations were performed with the R-Project free software Version 1.1.463.

3. Results

3.1. Strain Isolation, Morphological Characterization, and Biochemical Identification

In total, 58 rhizobacterial strains were obtained from the rhizoplane and endorhizosphere of casava roots, and most of them were grown on different media (Table 1 and Table 2). Based on the colony morphology on NA, AMA, KAB, and EMB media, all isolates had different morphological characteristics, including round-to-irregular colonies with flat and raised elevations, smooth and irregular surfaces, and diversity of size (Table 1 and Table 2). Several isolates produced pigments, with colonies that were white, off-white, milky white, purple, pink-purple, reddish, and yellowish in color, but some isolates did not produce any pigmentation (Table 1 and Table 2). All isolates reacted negatively to Gram staining but reacted positively and negatively to the catalase and oxidase tests, respectively (Table 1 and Table 2). VITEK® 2 identification showed that the isolates were mainly the members of genus Achromobacter, Acinetobacter, Aeromonas, Buttiauxella, Cronobacter, Klebsiella, Ochrobactrum, Pluralibacter, Pseudomonas, Rhizobium, Serratia, and Sphingomonas (Table 1 and Table 2). The probability levels of discrimination for all strains were ≥95% according to the VITEK® 2 system report.

3.2. Strains Diversity at the Beginning of the Drought Stress Assay (T0)

In total, 18 strains were isolated from the rhizoplane and endorhizosphere of cassava roots at T0, with 33.3% and 66.6% of bacteria isolated from roots of cp-c and cp-ds plants, respectively (Table 1). All isolates were identified at the species level using VITEK® 2. Concerning to identified isolates from the control plants, six rhizobacterial strains were the members of genus Pseudomonas, including four of Pseudomonas fluorescens, one of Pseudomonas putida, and one of Pseudomonas mendocina (Table 1). Contrariwise, 12 isolates were identified from the roots of cp-ds plants at the beginning of the assay. Members of the identified rhizobacteria in these plants were dominated by the genus Pseudomonas (50%), including species such as P. putida (n = 5) and P. fluorescens (n = 1). Moreover, species such as Sphingomonas paucimobilis (n = 4), Achromobacter xylosoxidans (n = 1), and Rhizobium radiobacter (n = 1) were also identified (Table 1). All the isolates produced colonies with different morphological characteristics (Table 1). According to the relative abundances, Pseudomonas and Sphingomonas were the predominant bacterial genera found in roots of cp-c and cp-ds plants at T0 (Figure 1).

3.3. Strains Diversity at the End of the Drought Stress Assay (T20)

In total, 40 rhizobacterial strains were isolated from the cassava roots of all plants after 20 days and identified (Table 2). The identified isolates produced colonies with different morphological characteristics (Table 2). In total, 21 identified strains were isolated from the cp-c plants, which underwent daily irrigation (Table 2). Bacterial species such as Acinetobacter baumannii (n = 1), A. xylosoxidans (n = 1), Aeromonas salmonicida (n = 1), Buttiauxella agrestis (n = 1), Cronobacter sakazakii (n = 1), Klebsiella pneumoniae (n = 1), Pluralibacter gergoviae (n = 1), P. putida (n = 1), P. fluorescens (n = 2), Pseudomonas stutzeri (n = 2), R. radiobacter (n = 4), Serratia marcescens (n = 1), and S. paucimobilis (n = 4) were identified in the root of the control plants after 20 days with daily irrigation (Table 2). By contrast, 19 isolates were isolated from roots of non-irrigated plants and then identified (Table 2). Several rhizobacterial species, including Ochrobactrum anthropi (n = 1), Pseudomonas luteola (n = 1), P. mendocina (n = 1), P. putida (n = 6), P. stutzeri (n = 2), R. radiobacter (n = 5), and S. paucimobilis (n = 4) were identified in cp-ds plants exposed to drought stress (Table 2). In this respect, Pseudomonas, Rhizobium, and Sphingomonas were the predominant bacterial genera found in the roots of the control cassava plants and plants exposed to drought stress (Figure 1).

3.4. Comparison of Strain Diversity for cp-c and cp-ds Plants

When populations of isolates were analyzed for the roots of cp-c and cp-ds plants, a higher diversity of rhizobacteria were found (Figure 1). First, a wider diversity of bacterial species and higher abundance were found at T20 than T0 for control plants (Table 3). The analysis of alpha diversity through Shannon diversity index revealed a marked effect of irrigation on the Gram-negative endophytic bacterial community of cassava roots after 20 days (Table 3). Daily irrigation for 20 days resulted in a significant increase in the Shannon diversity index in control plants (Table 3). In this respect, all rhizobacterial strains isolated from cp-c plants at the beginning of assay belonged to Gammaproteobacteria (Pseudomonalaes) (Table 1 and Figure 1). In contrast, after 20 days with daily irrigation, the rhizobacterial community associated with the roots of these plants exhibited changes in the relative abundance of bacteria, as well as an increased diversity (Figure 1 and Table 3). In this respect, members belonging to Alphaproteobacteria (Rhizobiales and Sphingomonadales), Gammaproteobacteria (Enterobacteriales, Pseudomonalaes, and Aeromonadales), and Betaproteobacteria (Burkholderiales) were notably abundant at T20 for control plants (Table 2 and Figure 1). Meanwhile, the evenness index showed no significant difference when comparing the bacterial community of control plants at T0 and T20 (Table 3).
On the other hand, water deprivation also influenced the Gram-negative rhizobacterial community of cassava after 20 days of drought stress. Despite cp-ds plants showing no significant change in the diversity, an increase in the Shannon diversity index of rhizobacterial endophytes of cassava plants after 20 days under water-deficit conditions was observed (Table 3). In this respect, an increase in the abundance and species richness was detected at the end of the drought stress assay (Table 3 and Figure 1). However, the proportion between Gammaproteobacteria (Pseudomonalaes) and Alphaproteobacteria (Rhizobiales and Sphingomonadales) was conserved between T0 and T20 for the roots of cp-ds plants (Figure 1). In particular, a slight increase in the relative abundance of Rhizobium genus was observed at the end of the drought stress assay (Table 2 and Figure 1). Similar to control plants, the evenness index of the rhizobacteria community from cp-ds plants showed no significant difference between T0 and T20 (Table 3).
Finally, changes in the relative abundances of predominant bacterial genera and species diversity were observed in both groups of plants at T20 (Figure 1 and Table 3). In this respect, when the bacterial community associated with the roots of the control plants under full irrigation was compared to that of plants under drought conditions at the end of 20 days of treatment, a higher diversity of rhizobacteria was found in the cp-c roots (Figure 1 and Table 3). Here, A. baumannii, A. salmonicida, B. agrestis, C. sakazakii, K. pneumoniae, P. gergoviae, and S. marcescens were bacterial species exclusively found in control plants at T20, whereas O. anthropi and P. luteola were exclusive rhizobacterial species of plants submitted to drought conditions (Figure 1 and Figure 2).

4. Discussion

Rhizobacteria can contribute to the adaptation of the plants to drought habitats [50]. In this study, we isolated rhizobacterial strains from the cassava M. esculenta Crantz MCOL1734, which is a variety with high tolerance capacity against drought. This study represents the first characterization of rhizobacterial community associated with cassava plants under drought stress conditions. In total, 58 Gram-negative rhizobacteria were isolated from the roots of the cassava plants exposed to daily irrigation and drought stress conditions. Previously, some studies have reported that Gram-negative rhizobacteria dominates the rhizosphere of several crop plants of agricultural importance [51,52]. According to the identification process with the VITEK® 2, we found that all the rhizobacterial strains isolated from cassava roots belonged to 12 genera: Achromobacter, Acinetobacter, Aeromonas, Buttiauxella, Cronobacter, Klebsiella, Ochrobactrum, Pluralibacter, Pseudomonas, Rhizobium, Serratia, and Sphingomonas. These genera include bacterial species previously reported as PGPRs. Several studies have showed that these taxa are dominant in the roots and tissue of plants, in most of cases as drought stress alleviators to ameliorate crop production [10,23,53]. The diversity of rhizobacteria found here was wider than that reported previously for several cassava varieties from India and China, which only included genera such as Enterobacter, Klebsiella, Pseudomonas, and Serratia [31,32].
We found that both water-deficit stress and daily irrigation changed the diversity, abundance, and richness of Gram-negative endophytic bacterial communities of cassava roots; however, changes in the evenness were not detected. In this respect, the Shannon diversity index revealed that the species diversity of bacterial isolates from roots of cp-c and cp-ds plants were higher at T20 compared to T0, although the difference was only significant in control plants. Meanwhile, the evenness index reflected the uniformity of bacterial species distribution at T0 and T20 for both cp-c and cp-ds plants, contrary to the trend found for species diversity (Table 3). Thus, the water deprivation and full irrigation had no significant effect on evenness, which suggests little effect on the relative abundances of either dominant or minor taxa. In particular, Pseudomonas, Rhizobium, and Sphingomonas were the predominant bacterial genera that we found in the roots of both cp-c and cp-ds plants of cassava. Pseudomonas spp. are characteristic in agricultural soils, being one of the most dominant genus commonly reported in many plant crops [54]. Several members of Pseudomonas genus have been widely studied as PGPRs that contribute to plant tolerance against abiotic stress [19,51,54] by producing a set of hormones involved in the growth and development of plants [26,53,55]. In this study, we observed that species such as P. fluorescens, P. putida, P. mendocina, P. stutzeri, and P. luteola prevailed in all plants throughout the treatment. A previous study reported that P. fluorescens produce higher amounts of 1-aminocyclopropane-1-carboxylate (ACC) deaminase that can promote the plant growth under drought stress conditions [20,50], whereas P. putida can induce the IAA production that plays a key role in both root and shoot development in plants [56]. Interestingly, we found that P. mendocina and P. stutzeri were species shared between control plants and plants exposed to drought stress, whereas P. luteola was isolated exclusively from plants submitted to drought conditions for 20 days. A previous study reported the role of these PGPR–mediating drought stress tolerance in plants through several mechanisms. The inoculation of lettuce under moderate and severe drought stress with P. mendocina significantly enhanced the phosphatase activity in roots, and the activities of nitrate reductase (N), peroxidase and catalase, and the proline accumulation in leaves [57]. Maize plants inoculated with Pseudomonas spp., including P. putida and P. stutzeri, developed protection against drought stress by reducing the activity of antioxidant enzymes [58]. Meanwhile, P. luteola played a key role during the development of roots in Malus domestica due to an increased production of IAA, siderophores, and biosurfactants, as well as the solubilization of organic and inorganic phosphorus [59]. In this respect, strains of Pseudomonas spp. isolated from cassava roots in this study could be considered excellent candidates to promote plant growth under drought stress conditions.
By contrast, orders such as Rhizobiales and Sphingomonadales include several PGPR that elicit plant drought tolerance [23]. In this respect, we detected the isolates of R. radiobacter and S. paucimobilis in the roots of cassava plants after 20 days of drought stress. Rhizobium spp. can produce CK that increase the development of principal and adventitious roots [60,61]. In addition, when plants of Phaseolus vulgaris were inoculated with Rhizoium spp. overexpressing the trehalose-6-phosphate synthase gene, an increase in the drought tolerance was observed [62]. Regarding Sphingomonas genus, some species have the capacity to grow around the root zone and promote the plant growth by producing primary metabolites [63]. Sphingomonas spp. synthesize siderophores that favor and promote the absorption of minerals and other ions by plants [64]. The inoculation of Dendrobium officinale with S. paucimobilis promoted the growth of seedlings through a combination of phytohormones and nitrogen fixation [63]. Moreover, Arabidopsis thaliana plants under drought stress increased the growth rate when they were inoculated with strains of Shingomonas sp. [65]. Therefore, the rhizobacterial strains of R. radiobacter and S. paucimobilis identified in this study have a great potential as PGPR strains that could contribute to plant tolerance against drought stress conditions.
Furthermore, we also identified other PGPRs genera such as Acinetobacter, Achromobacter, Aeromonas, Klebsiella, Serratia, and Ochrobactrum, but they were less abundant in the cassava roots. According to previous reports, several species of these rhizobacterial genera can induce the increase in plant growth and resistance to abiotic stresses through various mechanisms [20]. In this respect, Acinetobacter spp. have played an essential role in the alleviation of drought stress in the plants of Vigna radiata and Vitis vinifera through the production of IAA [66]. Likewise, strains of Serratia sp. induced drought tolerance in cucumber plants and promoted growth through IAA, ACC deaminase activity, and production of CK, siderophore production, and hydrogen cyanide [67]. Achromobacter spp. alleviated the adverse effects caused for abiotic stresses, including drought and oxidative stress, in tomato and peppers plants [68]. Thus, the inoculation of plants with Achromobacter sp. increased the biomass and significantly contributed to the reduction of ethylene levels in the stressed plants [68]. Finally, Ochrobactrum spp. strains grow in environments where water availability is a limiting factor [69]. This bacterial genus is involved in the release of organic acids and the efflux of hydrogen peroxide, which is a way of drought tolerance in plants [50]. Interestingly, we found that O. anthropi is exclusively present in cassava roots under water-deficit conditions, further suggesting that this rhizobacterial species is contributing to enhancing the tolerance to drought stress in cassava plants.

5. Conclusions

This study’s results showed that the Gram-negative rhizobacteria community was associated with M. esculenta Crantz MCOL1734 using culture-dependent approaches. A wider diversity of the root-associated bacterial microbiome was found in these plants. Our results demonstrate that the water-deficit stress clearly influences the species diversity of rhizobacterial community associated with cassava. In this respect, the water deprivation leads to changes in the diversity, relative abundances, and species richness of Gram-negative endophytic bacterial communities associated with cassava roots; however, no significant effect on evenness index of rhizobacterial species was found after drought stress assay. Moreover, a great diversity of bacterial species reported as PGPR species in previous studies were isolated from the roots of cassava plants under drought stress conditions and then identified. The rhizobacterial strains could improve the resistance and tolerance of cassava plants to drought throughout water stress while maintaining their viability. The cassava roots constitute a great reservoir of potential Gram-negative rhizobacteria with remarkable biotechnological applications. These strains can be used for including plant inoculation or development of bio-inoculants to improve the drought tolerance of plant crops under water-deficit conditions.

Author Contributions

Conceptualization, A.R.C.-D. and I.D.O.-I.; methodology, T.Z. and D.M.G.; formal analysis, T.Z., D.M.G., A.R.C.-D. and I.D.O.-I.; investigation, T.Z., D.M.G., A.R.C.-D. and I.D.O.-I.; resources, A.R.C.-D. and I.D.O.-I.; data curation, T.Z. and I.D.O.-I.; writing—original draft preparation, T.Z. and I.D.O.-I.; writing—review and editing, I.D.O.-I.; visualization, T.Z. and I.D.O.-I.; supervision, A.R.C.-D. and I.D.O.-I.; project administration, A.R.C.-D.; funding acquisition, A.R.C.-D. All authors have read and agreed to the published version of the manuscript.

Funding

The Dirección General de Investigaciones of Universidad Santiago de Cali under grant number DGI: 511-621117-C7 funded this research.

Acknowledgments

The authors thank Luis Augusto Becerra who is the leader of Cassava Program at International Center for Tropical Agriculture (CIAT) for supporting the execution of this study, German Patiño Romero for technical assistance, and Sandra Rivera Sanchez for her technical support at the laboratory. This research has been funded by Dirección General de Investigaciones of Universidad Santiago de Cali under call No. 01-2021.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The relative abundances of rhizobacterial species isolated from the cassava roots of control plants (cp-c) and plants exposed to drought stress (cp-ds), at the beginning (T0) and after 20 (T20) days of drought stress assay.
Figure 1. The relative abundances of rhizobacterial species isolated from the cassava roots of control plants (cp-c) and plants exposed to drought stress (cp-ds), at the beginning (T0) and after 20 (T20) days of drought stress assay.
Diversity 13 00366 g001
Diversity 13 00366 g002 550
Figure 2. Distribution of the number of rhizobacterial isolated from cassava roots. The Venn diagram illustrates the number of bacteria isolated from cassava roots of control plants (cp-c) and plants exposed to drought stress (cp-ds), at the beginning (T0) and after 20 (T20) days of drought stress.
Figure 2. Distribution of the number of rhizobacterial isolated from cassava roots. The Venn diagram illustrates the number of bacteria isolated from cassava roots of control plants (cp-c) and plants exposed to drought stress (cp-ds), at the beginning (T0) and after 20 (T20) days of drought stress.
Diversity 13 00366 g002
Table
Table 1. Biochemical analysis of the endophytic bacterial isolates of cassava roots at the beginning of drought stress assay (T0).
Table 1. Biochemical analysis of the endophytic bacterial isolates of cassava roots at the beginning of drought stress assay (T0).
Plant IDStrain IDGram ReactionColony Characteristics at Several Culture MediaBiochemical Analysis
NAAMAKABEMBCatalase TestOxidase TestIdentification (VITEK® 2)
cp-c-1cp-c-1.1–ve Diversity 13 00366 i001 Diversity 13 00366 i002 Diversity 13 00366 i003 Diversity 13 00366 i004++Pseudomonas putida
cp-c-1.2–ve Diversity 13 00366 i005 Diversity 13 00366 i006 Diversity 13 00366 i007 Diversity 13 00366 i008++Pseudomonas fluorescens
cp-c-1.3–ve Diversity 13 00366 i009 Diversity 13 00366 i010 Diversity 13 00366 i011 Diversity 13 00366 i012++Pseudomonas fluorescens
cp-c-2cp-c-2.1–ve Diversity 13 00366 i013 Diversity 13 00366 i014 Diversity 13 00366 i015 Diversity 13 00366 i016++Pseudomonas fluorescens
cp-c-2.2–ve Diversity 13 00366 i017 Diversity 13 00366 i018 Diversity 13 00366 i019 Diversity 13 00366 i020++Pseudomonas fluorescens
cp-c-2.3–ve Diversity 13 00366 i021 Diversity 13 00366 i022 Diversity 13 00366 i023 Diversity 13 00366 i024++Pseudomonas mendocina
cp-ds-1cp-ds-1.1–ve Diversity 13 00366 i025 Diversity 13 00366 i026 Diversity 13 00366 i027 Diversity 13 00366 i028++Pseudomonas putida
cp-ds-1.2–ve Diversity 13 00366 i029 Diversity 13 00366 i030 Diversity 13 00366 i031 Diversity 13 00366 i032++Pseudomonas putida
cp-ds-1.3–ve Diversity 13 00366 i033 Diversity 13 00366 i034 Diversity 13 00366 i035 Diversity 13 00366 i036++Pseudomonas fluorescens
cp-ds-2cp-ds-2.1–ve Diversity 13 00366 i037 Diversity 13 00366 i038 Diversity 13 00366 i039 Diversity 13 00366 i040++Pseudomonas putida
cp-ds-2.2–ve Diversity 13 00366 i041 Diversity 13 00366 i042 Diversity 13 00366 i043 Diversity 13 00366 i044++Pseudomonas putida
cp-ds-2.3–ve Diversity 13 00366 i045 Diversity 13 00366 i046 Diversity 13 00366 i047 Diversity 13 00366 i048++Pseudomonas putida
cp-ds-3cp-ds-3.1–ve Diversity 13 00366 i049 Diversity 13 00366 i050 Diversity 13 00366 i051 Diversity 13 00366 i052++Achromobacter xylosoxidans
cp-ds-3.2–ve Diversity 13 00366 i053 Diversity 13 00366 i054 Diversity 13 00366 i055 Diversity 13 00366 i056++Sphingomonas paucimobilis
cp-ds-4cp-ds-4.1–ve Diversity 13 00366 i057 Diversity 13 00366 i058 Diversity 13 00366 i059 Diversity 13 00366 i060++Sphingomonas paucimobilis
cp-ds-4.2–ve Diversity 13 00366 i061 Diversity 13 00366 i062 Diversity 13 00366 i063 Diversity 13 00366 i064++Sphingomonas paucimobilis
cp-ds-5cp-ds-5.1–ve Diversity 13 00366 i065 Diversity 13 00366 i066 Diversity 13 00366 i067 Diversity 13 00366 i068++Sphingomonas paucimobilis
cp-ds-5.2–ve Diversity 13 00366 i069 Diversity 13 00366 i070 Diversity 13 00366 i071 Diversity 13 00366 i072++Rhizobium radiobacter
Abbreviations: NA, nutrient agar; AMA, Ashby’s mannitol agar; KAB, King Agar B; EMB, eosin methylene blue agar.
Table
Table 2. Cellular and colony characteristics, as well as biochemical analysis of the endophytic bacterial isolates of cassava roots after 20 days of drought stress assay (T20).
Table 2. Cellular and colony characteristics, as well as biochemical analysis of the endophytic bacterial isolates of cassava roots after 20 days of drought stress assay (T20).
Plant IDStrain IDGram ReactionColony Characteristics at Several Culture MediaBiochemical Analysis
NAAMAKABEMBCatalase TestOxidase TestIdentification (VITEK® 2)
cp-c-1cp-c-1.4–ve Diversity 13 00366 i073 Diversity 13 00366 i074 Diversity 13 00366 i075 Diversity 13 00366 i076++Rhizobium radiobacter
cp-c-1.5–ve Diversity 13 00366 i077 Diversity 13 00366 i078 Diversity 13 00366 i079 Diversity 13 00366 i080+–Sphingomonas paucimobilis
cp-c-1.6–ve Diversity 13 00366 i081 Diversity 13 00366 i082 Diversity 13 00366 i083 Diversity 13 00366 i084+–Sphingomonas paucimobilis
cp-c-1.7–ve Diversity 13 00366 i085 Diversity 13 00366 i086 Diversity 13 00366 i087 Diversity 13 00366 i088–+Cronobacter sakazakii
cp-c-1.8–ve Diversity 13 00366 i089 Diversity 13 00366 i090 Diversity 13 00366 i091 Diversity 13 00366 i092++Rhizobium radiobacter
cp-c-1.9–ve Diversity 13 00366 i093 Diversity 13 00366 i094 Diversity 13 00366 i095 Diversity 13 00366 i096++Pluralibacter gergoviae
cp-c-1.10–ve Diversity 13 00366 i097 Diversity 13 00366 i098 Diversity 13 00366 i099 Diversity 13 00366 i100-+Serratia marcescens
cp-c-1.11–ve Diversity 13 00366 i101 Diversity 13 00366 i102 Diversity 13 00366 i103 Diversity 13 00366 i104++Pseudomonas putida
cp-c-1.12–ve Diversity 13 00366 i105 Diversity 13 00366 i106 Diversity 13 00366 i107 Diversity 13 00366 i108–+Klebsiella pneumoniae
cp-c-1.13–ve Diversity 13 00366 i109 Diversity 13 00366 i110 Diversity 13 00366 i111 Diversity 13 00366 i112++Pseudomonas stutzeri
cp-c-2cp-c-2.4–ve Diversity 13 00366 i113 Diversity 13 00366 i114 Diversity 13 00366 i115no growth++Pseudomonas stutzeri
cp-c-2.5–ve Diversity 13 00366 i116 Diversity 13 00366 i117 Diversity 13 00366 i118 Diversity 13 00366 i119++Acinetobacter baumannii
cp-c-2.6–ve Diversity 13 00366 i120 Diversity 13 00366 i121 Diversity 13 00366 i122 Diversity 13 00366 i123++Rhizobium radiobacter
cp-c-2.7–ve Diversity 13 00366 i124 Diversity 13 00366 i125 Diversity 13 00366 i126 Diversity 13 00366 i127-+Achromobacter xylosoxidans
cp-c-2.8–ve Diversity 13 00366 i128 Diversity 13 00366 i129 Diversity 13 00366 i130 Diversity 13 00366 i131-+Sphingomonas paucimobilis
cp-c-2.9–ve Diversity 13 00366 i132 Diversity 13 00366 i133 Diversity 13 00366 i134 Diversity 13 00366 i135++Rhizobium radiobacter
cp-c-2.10–ve Diversity 13 00366 i136 Diversity 13 00366 i137 Diversity 13 00366 i138 Diversity 13 00366 i139++Sphingomonas paucimobilis
cp-c-2.11–ve Diversity 13 00366 i140 Diversity 13 00366 i141 Diversity 13 00366 i142no growth-+Buttiauxella agrestis
cp-c-2.12–ve Diversity 13 00366 i143 Diversity 13 00366 i144 Diversity 13 00366 i145 Diversity 13 00366 i146++Aeromonas salmonicida
cp-c-2.13–ve Diversity 13 00366 i147 Diversity 13 00366 i148 Diversity 13 00366 i149 Diversity 13 00366 i150++Pseudomonas fluorescens
cp-c-2.14–ve Diversity 13 00366 i151 Diversity 13 00366 i152 Diversity 13 00366 i153 Diversity 13 00366 i154++Pseudomonas fluorescens
cp-ds-1cp-ds-1.4–ve Diversity 13 00366 i155 Diversity 13 00366 i156 Diversity 13 00366 i157 Diversity 13 00366 i158++Pseudomonas putida
cp-ds-1.5–ve Diversity 13 00366 i159 Diversity 13 00366 i160 Diversity 13 00366 i161 Diversity 13 00366 i162++Pseudomonas putida
cp-ds-2cp-ds-2.4–ve Diversity 13 00366 i163 Diversity 13 00366 i164 Diversity 13 00366 i165 Diversity 13 00366 i166++Pseudomonas putida
cp-ds-2.5–ve Diversity 13 00366 i167 Diversity 13 00366 i168 Diversity 13 00366 i169 Diversity 13 00366 i170++Pseudomonas mendocina
cp-ds-2.6–ve Diversity 13 00366 i171 Diversity 13 00366 i172 Diversity 13 00366 i173 Diversity 13 00366 i174++Ochrobactrum anthropi
cp-ds-2.7–ve Diversity 13 00366 i175 Diversity 13 00366 i176 Diversity 13 00366 i177 Diversity 13 00366 i178++Sphingomonas paucimobilis
cp-ds-3cp-ds-3.3–ve Diversity 13 00366 i179 Diversity 13 00366 i180 Diversity 13 00366 i181 Diversity 13 00366 i182++Rhizobium radiobacter
cp-ds-3.4–ve Diversity 13 00366 i183 Diversity 13 00366 i184 Diversity 13 00366 i185 Diversity 13 00366 i186++Sphingomonas paucimobilis
cp-ds-3.5–ve Diversity 13 00366 i187 Diversity 13 00366 i188 Diversity 13 00366 i189 Diversity 13 00366 i190-+Pseudomonas stutzeri
cp-ds-4cp-ds-4.3–ve Diversity 13 00366 i191 Diversity 13 00366 i192 Diversity 13 00366 i193 Diversity 13 00366 i194++Pseudomonas putida
cp-ds-4.4–ve Diversity 13 00366 i195 Diversity 13 00366 i196 Diversity 13 00366 i197 Diversity 13 00366 i198-+Rhizobium radiobacter
cp-ds-4.5–ve Diversity 13 00366 i199 Diversity 13 00366 i200 Diversity 13 00366 i201 Diversity 13 00366 i202++Pseudomonas putida
cp-ds-4.6–ve Diversity 13 00366 i203 Diversity 13 00366 i204 Diversity 13 00366 i205 Diversity 13 00366 i206++Rhizobium radiobacter
cp-ds-4.7–ve Diversity 13 00366 i207 Diversity 13 00366 i208 Diversity 13 00366 i209 Diversity 13 00366 i210++Sphingomonas paucimobilis
cp-ds-5cp-ds-5.3–ve Diversity 13 00366 i211 Diversity 13 00366 i212 Diversity 13 00366 i213 Diversity 13 00366 i214++Pseudomonas putida
cp-ds-5.4–ve Diversity 13 00366 i215 Diversity 13 00366 i216 Diversity 13 00366 i217 Diversity 13 00366 i218++Pseudomonas luteola
cp-ds-5.5–ve Diversity 13 00366 i219 Diversity 13 00366 i220 Diversity 13 00366 i221 Diversity 13 00366 i222-+Rhizobium radiobacter
cp-ds-5.6–ve Diversity 13 00366 i223 Diversity 13 00366 i224 Diversity 13 00366 i225 Diversity 13 00366 i226++Sphingomonas paucimobilis
cp-ds-5.7–ve Diversity 13 00366 i227 Diversity 13 00366 i228 Diversity 13 00366 i229 Diversity 13 00366 i230++Rhizobium radiobacter
Abbreviations: NA, nutrient agar; AMA, Ashby’s mannitol agar; KAB, King agar B; EMB, eosin methylene blue agar.
Table
Table 3. Abundance, species richness, Shannon diversity index, and Pielou evenness index of the endophytic bacterial isolates identified in cassava control plants (cp-c) and cassava plants exposed to drought stress (cp-ds), at the beginning (T0) and after 20 days (T20).
Table 3. Abundance, species richness, Shannon diversity index, and Pielou evenness index of the endophytic bacterial isolates identified in cassava control plants (cp-c) and cassava plants exposed to drought stress (cp-ds), at the beginning (T0) and after 20 days (T20).
Cassava Plants TreatmentsAbundanceSpecies RichnessShannon Diversity Index (H′)p-Value 1Evennessp-Value 2
cp-c_T0630.8680.00190.7890.7070
cp-c_T2021132.3840.929
cp-ds_T01251.3500.28990.8400.9678
cp-ds_T201971.6630.855
1 Significance level for Shannon diversity index of rhizobacteria community of cp-c between T0 and T20 and cp-ds between T0 and T20. 2 Significance level for the Pielou evenness index of rhizobacteria community of cp-c between T0 and T20 and cp-ds between T0 and T20.
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Zapata, T.; Galindo, D.M.; Corrales-Ducuara, A.R.; Ocampo-Ibáñez, I.D. The Diversity of Culture-Dependent Gram-Negative Rhizobacteria Associated with Manihot esculenta Crantz Plants Subjected to Water-Deficit Stress. Diversity 2021, 13, 366. https://doi.org/10.3390/d13080366

AMA Style

Zapata T, Galindo DM, Corrales-Ducuara AR, Ocampo-Ibáñez ID. The Diversity of Culture-Dependent Gram-Negative Rhizobacteria Associated with Manihot esculenta Crantz Plants Subjected to Water-Deficit Stress. Diversity. 2021; 13(8):366. https://doi.org/10.3390/d13080366

Chicago/Turabian Style

Zapata, Tatiana, Diana Marcela Galindo, Alba Rocío Corrales-Ducuara, and Iván Darío Ocampo-Ibáñez. 2021. "The Diversity of Culture-Dependent Gram-Negative Rhizobacteria Associated with Manihot esculenta Crantz Plants Subjected to Water-Deficit Stress" Diversity 13, no. 8: 366. https://doi.org/10.3390/d13080366

APA Style

Zapata, T., Galindo, D. M., Corrales-Ducuara, A. R., & Ocampo-Ibáñez, I. D. (2021). The Diversity of Culture-Dependent Gram-Negative Rhizobacteria Associated with Manihot esculenta Crantz Plants Subjected to Water-Deficit Stress. Diversity, 13(8), 366. https://doi.org/10.3390/d13080366

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